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The nuclear pore complex (NPC) is a large protein assembly that mediates molecular trafficking between the cytoplasm and the nucleus. INM proteins are required to initiate interphase NPC assembly. Our data also suggest, for the first time, the importance of the INM as a seeding site for prepores during interphase NPC assembly. Tariquidar (XR9576) INTRODUCTION The eukaryotic genome is usually segregated from the cytoplasm by a pair of lipid bilayers referred to as the nuclear envelope (NE). The NE is made up of the outer nuclear membrane (ONM), the inner nuclear membrane (INM), and nuclear pore complexes (NPCs) that span both membranes. The ONM is usually continuous with the endoplasmic reticulum (ER) and shows ER-like properties such as the presence of bound ribosomes. In contrast, the INM contains a unique set of integral membrane proteins, which, in vertebrates, are connected to the nuclear lamina (Daigle egg extracts and high-resolution imaging showed that new NPCs assemble on the NE by a de novo mechanism in which the scaffold Nup107C160 complex is usually inserted into NPC assembly sites from both the cytoplasmic and nucleoplasmic sides of the NE (DAngelo egg extracts have revealed the molecular basis of this recruitment: Pom121 is usually retained in the same membrane vesicles as Ndc1 and interacts directly with components of the Nup107C160 complex and the Nup205C93 complex (the vertebrate homologue of the yeast Nup170p complex) (Antonin reported that Pom121 colocalized with the Nup107C160 complex on the inner, but not the outer, nuclear membrane in an early NPC assembly intermediate manipulated in egg extract (Fichtman I-BL21 and purified with Glutathione Sepharose 4B beads (17C0756; GE Healthcare). Because the GST-Pom121137C513 peptide became unpredictable during purification, a (His)6 tag was added to its C terminus, and it was isolated using Ni-NTA agarose (30210; Qiagen) prior Tariquidar (XR9576) to purification with Glutathione Sepharose 4B. GST-Pom121 peptideCcoated beads and GST-coated beads were incubated for 1 h with cytosol from HeLa cells, which was prepared Tariquidar (XR9576) based on the statement by Hawryluk-Gara (2005 ). Briefly, cells were hanging in lysis buffer (20 mM Tris-HCl [pH Tariquidar (XR9576) 7.5], 400 mM NaCl, 1 mM EDTA, 1% Triton Times-100, 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail [11873580001; Roche]), sonicated, and centrifuged at 15,000 for 30 min at 4C. Supernatants were diluted 3.75-fold in binding buffer, giving final concentrations of Triton X-100 and NaCl of 0.3% and 106 mM, respectively, and centrifuged at 15,000 for 15 min at 4C. GST-Pom121 fragments and GST-coated beads were incubated with the diluted supernatants, washed with binding buffer, eluted in sample buffer, and analyzed by Western blotting. To observe direct interactions, wild-type and NLS mutant GST-Pom121266C513Ccoated Glutathione Sepharose 4B beads were incubated for 1 h at 4C with recombinant importin , importin , or transportin. These proteins experienced all been purified as previously reported (Imamoto of a bleached NE surface spot (at), a nonbleached NE surface spot (bC cC c= 0 was plotted. Supplementary Material [Supplemental Materials] Click here to view. Acknowledgments We thank A. Miyawaki, T. Nagai, and R. Tsien for their gifts of Venus and SECFP. We also thank users of the Cellular Mechanics Lab for their helpful feedback. We are thankful to R. Nakazawa, Y. Ichikawa, and the Support Unit at the RIKEN BSI Research Resources Center for help with DNA sequencing. This work was supported by a MEXT grant-in-aid and from RIKEN Special Project Funding for Basic Science (Bioarchitect Project and Cellular System Project). Abbreviations used: BHKbaby hamster kidneyEGFPenhanced green fluorescent proteinERendoplasmic reticulumFGphenylalanine-glycineFRAPfluorescence recovery after photobleachingGFPgreen fluorescent proteinGSTglutathione S-transferaseGTPguanosine-5-triphosphateH2Bhistone 2BINMinner nuclear membraneLBRlamin W receptormAbmonoclonal antibodyNEnuclear envelopeNGSnormal goat serumNLSnuclear localization signalNPCnuclear pore complexNupnucleoporinONMouter nuclear membranePBSphosphate-buffered salinePEGpolyethylene glycolPom, pore membrane proteinRCC1regulator of chromosome condensation 1RNAiRNA interferenceSECFPsuper enhanced cyan fluorescent proteinsiRNAsmall interfering RNA Footnotes This article was published online IL-1A ahead of print in MBoC Tariquidar (XR9576) in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10-07-0641) on February 2, 2011. Recommendations Alber F, et al. The molecular architecture of the nuclear pore complex. Nature. 2007;450:695C701. [PubMed]Antonin W, Franz C, Haselmann U, Antony C, Mattaj IW. The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation. Mol Cell. 2005;17:83C92. [PubMed]Cronshaw JM, Krutchinsky AN, Zhang W, Chait BT, Matunis MJ. Proteomic analysis of the mammalian nuclear pore complex. J Cell Biol. 2002;158:915C927. [PMC free article] [PubMed]DAngelo MA, Anderson DJ, Richard At the, Hetzer MW. Nuclear pores form de novo from both sides of the nuclear envelope. Science. 2006;312:440C443. [PubMed]Daigle N, Beaudouin J, Hartnell T, Imreh G, Hallberg At the, Lippincott-Schwartz J, Ellenberg J. Nuclear pore complexes form immobile networks and have a very low turnover.

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